ABA mediates seasonal drought-induced citrate accumulation in citrus fruit through transcriptional and post-translational regulation of CsMADS1

Seasonal drought at fruit enlargement stage increases citrus fruit acidity, but the underlying mechanism remains incompletely understood. Here, CsMADS1, a MADS-box transcription factor, was identified as a positive regulator of abscisic acid (ABA)-induced citrate accumulation associated with seasonal drought in citrus fruit. CsMADS1 was upregulated by drought and ABA, and its expression was positively correlated with citrate content in citrus cultivars. Overexpression of CsMADS1 in citrus tissues enhanced citrate accumulation, whereas CsMADS1 silencing reduced it. CsMADS1 directly bound to the promoter of ANTHOCYANIN1 (CsAN1), a bHLH transcription factor involved in citrate transport, and activated its transcription. CsAN1 further activated CsPH8, a P_3A-ATPase proton pump gene, thereby contributing to vacuolar acidification. In addition, the ABA-responsive protein kinase CsSnRK2.6 interacted with and phosphorylated CsMADS1 at Ser¹⁴⁰. This phosphorylation enhanced CsMADS1-mediated transactivation of CsAN1 but was not strictly required for ABA responsiveness. Citrate measurements in citrus calli showed that CsSnRK2.6 promoted citrate accumulation mainly under ABA treatment and that the phosphomimetic CsMADS1^S140D variant showed stronger activity than CsMADS1. Together, these findings support a refined model in which ABA regulates CsMADS1 through both transcriptional induction and post-translational phosphorylation, leading to activation of the CsAN1–CsPH8 pathway and citrate accumulation. Our findings provide insights into ABA-mediated citrate accumulation and suggest a potential strategy for improving citrus fruit quality.